Solution structures of the N-terminal domain of yeast calmodulin: Ca2+-dependent conformational change and its functional implication

We have determined solution structures of the N-terminal half domain (N-domain) of yeast calmodulin (YCM0-N, residues 1-77) in the apo and Ca(2+)-saturated forms by NMR spectroscopy. The Ca(2+)-binding sites of YCM0-N consist of a pair of helix-loop-helix motifs (EF-hands), in which the loops are linked by a short beta-sheet. The binding of two Ca(2+) causes large rearrangement of the four alpha-helices and exposes the hydrophobic surface as observed for vertebrate calmodulin (CaM). Within the observed overall conformational similarity in the peptide backbone, several significant conformational differences were observed between the two proteins, which originated from the 38% disagreement in amino acid sequences. The beta-sheet in apo YCM0-N is strongly twisted compared with that in the N-domain of CaM, while it turns to the normal more stable conformation on Ca(2+) binding. YCM0-N shows higher cooperativity in Ca(2+) binding than the N-domain of CaM, and the observed conformational change of the beta-sheet is a possible cause of the highly cooperative Ca(2+) binding. The hydrophobic surface on Ca(2+)-saturated YCM0-N appears less flexible due to the replacements of Met51, Met71, and Val55 in the hydrophobic surface of CaM with Leu51, Leu71, and Ile55, which is thought to be one of reasons for the poor activation of target enzymes by yeast CaM.     

Evaluation of nine sets of PCR primers in the RNA dependent RNA polymerase region for detection and differentiation of members of the family Caliciviridae, Norwalk virus and Sapporo virus

Norwalk virus and Sapporo virus were approved as type species of the genus "Norwalk-like viruses" and the genus "Sapporo-like viruses," respectively, in the family Caliciviridae. A total of 116 stool specimens containing Norwalk virus (NV) or Sapporo virus (SV) were tested by RT-PCR and Southern hybridization to evaluate nine sets of PCR primers and seven internal oligonucleotide probes in the RNA dependent RNA polymerase region of NV and SV for detection and differentiation of viruses in the NV and SV. Fifty-five stool samples were collected from 11 outbreaks of NV and/or SV gastroenteritis in an infant home, where residents were infants under 2 years of age, in Sapporo, Japan. Sixty specimens were obtained in Sapporo from sporadic cases in children, mainly under 6 years of age, of acute gastroenteritis due to small round structured viruses detected by EM. There is no single primer pair to detect all NV and SV, and at least three primer pairs, G1 set, G2 set and Sapp35/Sapp36, are required to detect viruses in the NV and SV clades. Many NV and SV strains were successfully classified into one of the NV/genogroup I, NV/genogroup II and SV by single-round RT-PCR and Southern hybridization. The new detection method for SV reported in this study combined with those for NV previously reported may elucidate the importance of Norwalk virus and Sapporo virus as a cause of viral gastroenteritis in all age groups in the world.     

Absolute configuration and tautomeric structure of xylindein, a blue-green pigment of Chlorociboria species

The S absolute configuration of both chiral centers of xylindein was assigned using X-ray crystallographic heavy atom analysis after its conversion to a synthetic derivative. Crystallographic analysis of xylindein crystallized with phenols revealed that the proposed structure is the proper tautomer in the crystals.     

Molecular cloning and expression of the mouse N-acetylneuraminic acid 9-phosphate synthase which does not have deaminoneuraminic acid (KDN) 9-phosphate synthase activity

A cDNA of the mouse homologue of Escherichia coli N-acetylneuraminic acid (Neu5Ac) synthase (neuB gene product) was cloned by the PCR-based method. The mouse homologue consists of 359 amino acids, and the cDNA sequence displays 33% identity to that of the E. coli Neu5Ac synthase. The recombinant mouse homologue which is transiently expressed in HeLa cells does not exhibit the Neu5Ac synthase activity, which catalyzes condensation of phosphoenolpyruvate (PEP) and N-acetylmannosamine (ManNAc) to synthesize Neu5Ac, but the Neu5Ac 9-phosphate (Neu5Ac-9-P) synthase activity, which catalyzes condensation of PEP and ManNAc 6-phosphate (ManNAc-6-P) to synthesize Neu5Ac-9-P. Thus, the mouse homologue of E. coli Neu5Ac synthase is the Neu5Ac-9-P synthase. The Neu5Ac-9-P synthase is a cytosolic enzyme and ubiquitously distributed in mouse various tissues. Notably, the Neu5Ac-9-P synthase can not catalyze the synthesis of deaminoneuraminic acid (KDN) or KDN-9-P from PEP and Man or ManNAc-6-P, thus suggesting that the enzyme is not involved in the synthesis of KDN. This is consistent with the previous observation that only a very low activity to synthesize KDN is found in mouse B16 cells [Angata, T., et al. (1999) Biochem. Biophys. Res. Commun. 261, 326-331].     

Diapause and survival in winter in two species of predatory bugs,Orius sauteri and O. minutus

The cause of differences in overwintering success between the sexes in Orius sauteri (Poppius) and O. minutus (Linnaeus) (Heteroptera: Anthocoridae) was investigated in the laboratory. The survival rate of adults was examined in a screen house outdoors during the winters of 1995–1996 and 1996–1997. None of the males of either species survived until spring in either year. However, 63.9% and 40.5% of the females in O. sauteri and O. minutus, respectively, survived the winter in 1995–1996, and 54.5% and 43.2%, respectively, in 1996–1997. Most of the males died by early winter. In both species, the adult females reared under short days (= L11:D13) survived for a long period at 5 or 0°C, while the males reared under the same photoperiod died shortly after transfer to 5 or 0°C from 22°C. The males and females kept at same temperature conditions under long days (= L16:D8) died early. When nymphs were kept under long days at 22°C, the lipid content in the female and male adults of O. sauteri was 27.9% and 17.7%, respectively. When nymphs were kept under short days, their lipid content was significantly higher (41.1%) than that of those reared under long days for females, but lipid content was comparable in the males regardless of photoperiod (15.6%). In O. minutus, the mean lipid content was 27.2% in females and 19.0% in males under long days at 22°C, and 40.8% in females and 19.8% in males under short days. Thus, a large amount of lipid was accumulated only in females kept under short days in both species. These results suggest that short days induced diapause only in adult females but not in males. Due to the lack of diapause and shortage of lipid accumulation, males may not be able to survive the winter.     

Visualization of the Dynamics of Synaptic Vesicle and Plasma Membrane Proteins in Living Axons

Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles. However, their transporting vesicles have not yet been identified. We have successfully visualized the transporting vesicles of plasma membrane proteins, synaptic vesicle proteins, and the trans-Golgi network residual proteins in living axons at high resolution using laser scan microscopy of green fluorescent protein-tagged proteins after photobleaching. We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement. These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.     

More than a 600-fold variation in nitrogen dioxide assimilation among 217 plant taxa

Assimilation of nitrogen dioxide in response to fumigation with ¹⁵N-labelled nitrogen dioxide was studied in 217 plant taxa. The taxa included 50 wild herbaceous plants collected from roadsides (42 genera, 15 families), 60 cultivated herbaceous plants (55 genera, 30 families) and 107 cultivated woody plants (74 genera, 45 families). Two parameters, the 'NO₂-N content', or NO₂-derived reduced nitrogen content in fumigated plant leaves (mg N g^–1 dry weight), and the 'NO₂-utilization index', or percentage of the NO₂-derived reduced nitrogen in the total reduced nitrogen, were determined. The NO₂-N content differed 657-fold between the highest ( Eucalyptus viminalis ; 6·57) and lowest ( Tillandsia ionantha and T. caput-medusae ; 0·01) values in the 217 taxa; 62-fold in a family (Theaceae) and 26-fold in a species ( Solidago altissima ). Nine species had NO₂-utilization indices greater than 10%, of which Magnolia kobus , Eucalyptus viminalis , Populus nigra , Nicotiana tabacum and Erechtites hieracifolia had NO₂-N contents > 4·9. These plants can be considered 'NO₂-philic' because in them NO₂-nitrogen has an important function(s). The Compositae and Myrtaceae had high values for both parameters, whereas the monocots and gymnosperms had low ones. These findings suggest that the metabolic pathway of NO₂-nitrogen differs among plant species. The information presented here will be useful for creating a novel vegetation technology to reduce the atmospheric concentration of nitrogen dioxide.     

The MAP kinase kinase kinase MLK2 co-localizes with activated JNK along microtubules and associates with kinesin superfamily motor KIF3.

The MLK (mixed lineage) ser/thr kinases are most closely related to the MAP kinase kinase kinase family. In addition to a kinase domain, MLK1, MLK2 and MLK3 each contain an SH3 domain, a leucine zipper domain and a potential Rac/Cdc42 GTPase-binding (CRIB) motif. The C-terminal regions of the proteins are essentially unrelated. Using yeast two-hybrid analysis and in vitro dot-blots, we show that MLK2 and MLK3 interact with the activated (GTP-bound) forms of Rac and Cdc42, with a slight preference for Rac. Transfection of MLK2 into COS cells leads to strong and constitutive activation of the JNK (c-Jun N-terminal kinase) MAP kinase cascade, but also to activation of ERK (extracellular signal-regulated kinase) and p38. When expressed in fibroblasts, MLK2 co-localizes with active, dually phosphorylated JNK1/2 to punctate structures along microtubules. In an attempt to identify proteins that affect the activity and localization of MLK2, we have screened a yeast two-hybrid cDNA library. MLK2 and MLK3 interact with members of the KIF3 family of kinesin superfamily motor proteins and with KAP3A, the putative targeting component of KIF3 motor complexes, suggesting a potential link between stress activation and motor protein function.     

Contrasting Effects of the Cytotoxic Anticancer Drug Gemcitabine and the EGFR Tyrosine Kinase Inhibitor Gefitinib on NK Cell-Mediated Cytotoxicity via Regulation of NKG2D Ligand in Non-Small-Cell Lung Cancer Cells

IntroductionSeveral cytotoxic anticancer drugs inhibit DNA replication and/or mitosis, while EGFR tyrosine kinase inhibitors inactivate EGFR signalling in cancer cell. Both types of anticancer drugs improve the overall survival of the patients with non-small-cell lung cancer (NSCLC), although tumors often become refractory to this treatment. Despite several mechanisms by which the tumors become resistant having been described the effect of these compounds on anti-tumor immunity remains largely unknown.MethodsThis study examines the effect of the cytotoxic drug Gemcitabine and the EGFR tyrosine kinase inhibitor Gefitinib on the expression of NK group 2 member D (NKG2D) ligands as well as the sensitivity of NSCLC cells to the NK-mediated lysis.ResultsWe demonstrate that Gemcitabine treatment leads to an enhanced expression, while Gefitinib downregulated the expression of molecules that act as key ligands for the activating receptor NKG2D and promote NK cell-mediated recognition and cytolysis. Gemcitabine activated ATM and ATM- and Rad-3-related protein kinase (ATR) pathways. The Gemcitabine-induced phosphorylation of ATM as well as the upregulation of the NKG2D ligand expression could be blocked by an ATM-ATR inhibitor. In contrast, Gefitinib attenuated NKG2D ligand expression. Silencing EGFR using siRNA or addition of the PI3K inhibitor resulted in downregulation of NKG2D ligands. The observations suggest that the EGFR/PI3K pathway also regulates the expression of NKG2D ligands. Additionally, we showed that both ATM-ATR and EGFR regulate MICA/B via miR20a.ConclusionIn keeping with the effect on NKG2D expression, Gemcitabine enhanced NK cell-mediated cytotoxicity while Gefitinib attenuated NK cell killing in NSCLC cells.